feasibility of biohydrogen production from cheese whey using a uasb reactor: links between microbial...

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Feasibility of biohydrogen production from cheesewhey using a UASB reactor: Links between microbialcommunity and reactor performance

E. Castelloa,*, C. Garcıa y Santosa, T. Iglesiasb, G. Paolinob, J. Wenzelb,L. Borzacconia, C. Etchebehereb

aChemical Engineering Institute, School of Engineering, University of the Republic, Herrera y Reissig 565, Montevideo, UruguaybMicrobiology Department, School of Science and School of Chemistry, University of the Republic, General Flores 2124, Montevideo, Uruguay

a r t i c l e i n f o

Article history:

Received 9 March 2009

Received in revised form

7 May 2009

Accepted 14 May 2009

Available online 21 June 2009

Keywords:

Hydrogen production

UASB

Lactose fermentation

Cheese whey

* Corresponding author. Facultad de IngenierTel.: þ5982 711 08 71, þ5982 711 44 78; fax: þ

E-mail address: [email protected] (E.0360-3199/$ – see front matter ª 2009 Interndoi:10.1016/j.ijhydene.2009.05.060

a b s t r a c t

The present study examines the feasibility of producing hydrogen by dark fermentation

using unsterilised cheese whey in a UASB reactor. A lab-scale UASB reactor was operated

for more than 250 days and unsterilised whey was used as the feed. The evolution of the

microbial community was studied during reactor operation using molecular biology tools

(T-RFLP, 16S rRNA cloning library and FISH) and conventional microbiological techniques.

The results showed that hydrogen can be produced but in low amounts. For the highest

loading rate tested (20 gCOD/L.d), hydrogen production was 122 mL H2/L.d. Maintenance of

low pH (mean¼ 5) was insufficient to control methanogenesis; methane was produced

concomitantly with hydrogen, suggesting that the methanogenic biomass adapted to the

low pH conditions. Increasing the loading rate to values of 2.5 gCOD/gVSS.d favoured

hydrogen production in the reactor. Microbiological studies showed the prevalence of

fermentative organisms from the genera Megasphaera, Anaerotruncus, Pectinatus and Lacto-

bacillus, which may be responsible for hydrogen production. However, the persistence of

methanogenesis and the presence of other fermenters, not clearly recognised as hydrogen

producers indicates that competition for the substrate may explain the low hydrogen

production.

ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights

reserved.

1. Introduction production by dark fermentation is to block consumption by

Hydrogen is a clean energy carrier that possesses a high

energy yield (122 kJ g�1) and does not contribute to the

greenhouse effect. There are many different hydrogen

production processes: electrolysis, natural gas reforming and

biological processes, such as dark fermentation of carbohy-

drate-rich substrates [1,2]. The main strategy for hydrogen

ıa, Universidad de la Rep5982 710 74 37.

Castello).ational Association for H

methanogens and to select for high-yield hydrogen producers.

There are several genera of Bacteria known to produce

hydrogen by dark fermentation. Among them, members of the

Clostridium genera have the highest theoretical yield (4 mol H2-

mol hexose�1) [3]; however, the yields reported in the litera-

ture are lower [2,4]. This could be due to the incorporation of

substrate by the biomass, the production of fermentation

ublica, Herrera y Reisig 565, CP.11300, Montevideo, Uruguay.

ydrogen Energy. Published by Elsevier Ltd. All rights reserved.

mailto:[email protected]

http://www.elsevier.com/locate/he

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 5 6 7 4 – 5 6 8 2 5675

products other than acetate and the consumption of hydrogen

by methanogens [2,5].

Biological hydrogen production has the advantage of

a low energy demand compared to other technologies [6].

The possibility of using organic wastes as substrates makes

the process even more attractive. Cheese whey is a by-

product generated during cheese manufacturing. The main

components are lactose (70–72% dried extract), proteins (8–

10%), and mineral salts (12–15% of dried extract) [7]. Proper

management of cheese whey is important due to stricter

legislation that does not permit its land disposal without

prior treatment, as well as economic reasons that force its

valorisation. Cheese whey has an elevated carbohydrate

(lactose) concentration and a low buffer capacity. Its treat-

ment in a conventional anaerobic reactor frequently leads to

acidification and inhibition of methanogenic activity [8].

These characteristics make this by-product a good substrate

for biohydrogen production. Hydrogen production should be

followed by a stage of methane production in order to

recover all the energy content of the cheese whey and

reduce the COD. There has been some experience working

with dry whey powder as a substrate for biohydrogen

production in continuous and batch modes [9,10], but the

ability to produce hydrogen using raw whey remains to be

evaluated.

Various technologies for hydrogen production can be

found in the literature, all of them at lab-scale: continuous

stirred tank reactors [9,11], sequencing batch reactors [12] and

upflow sludge bed reactors [13,14]. The operational conditions

that optimise the hydrogen production process have not been

completely defined, but pH and hydraulic retention time (HRT)

have been reported as the most important parameters to

control. To optimise the hydrogen production, reactors should

be operated at pH 5.5 with HRT between 8 and 12 h [2,15–17];

however, there are also reports of a wider range of pH for

optimum operation, between 4.5 and 6.5 [6].

Most previous investigations were carried out using

synthetic wastewater. Therefore, more information is needed

on the applicability of the hydrogen production process to

industrial wastewater due to the possible presence of unde-

sirable microorganisms.

The objectives of this work were to study the applicability

of dark fermentation for hydrogen production in a UASB

reactor fed with raw, unsterilised cheese whey, and to eval-

uate the effect of increasing the organic loading rate. The

evolution of the microbial community was linked to the

reactor operational data to better understand the process.

2. Materials and methods

2.1. Substrate

Cheese whey was obtained from a local cheese production

factory. It was received from the factory once per week and

stored at 4 �C until used. The average composition of the

cheese whey was as follows: COD 67,000 mg/L (standard

deviation 6000 mg/L, 66 samples); total nitrogen 1335 mgN/L;

total phosphorus 310 mg/L; and pH 4.7 (standard deviation 0.9,

66 samples). Prior to being fed into the reactor, the whey was

diluted to a COD concentration of 10,000 mg/L and supple-

mented with NaHCO3 (0.2 gNaHCO3/gCOD). The addition of

NaHCO3 was started at day 5 of operation after observing

a significant decrease in the pH of the reactor.

2.2. Seed sludge

The seed sludge was obtained from an acidogenic lab-scale

reactor fed with glucose that had been in operation for 3

months. No pre-treatment of the sludge was carried out prior

to its inoculation in the reactor.

2.3. The reactor system

A laboratory-scale UASB reactor (working volume 4.6 L, height

54 cm) with 4 sampling points along its height was used for

biohydrogen production. The reactor was placed in a 30 �C

thermostatic chamber, and biogas production was measured

with a water displacement meter.

The reactor was started with a hydraulic residence time

(HRT) of 24 h and a COD concentration of 10,000 mg/L. The

COD level was kept constant during the operation. The

organic loading rate under those conditions was 10 gCOD/L.d.

To promote the elimination of methanogenesis, the HRT was

reduced to 12 h on day 56. From that point on, the organic

loading rate was 20 gCOD/L.d. Other changes in the hydraulic

residence time were due to operating problems.

2.4. Analytical methods

The determinations of chemical oxygen demand (COD), total

suspended solids (TSS) and volatile suspended solids (VSS)

were carried out according to standard methods [18].

Hydrogen and methane were determined by gas chromatog-

raphy (Chromatograph SRI 8610) using a molecular sieve 13�column (Chrompack) and TCD detector. Volatile fatty acids

(VFA) were determined by HPLC with the following operating

conditions: polymeric column ORH-801, UV detector (Shi-

madzu 10AD) at 210 nm, mobile phase H2SO4 (0.005 M), a flow

rate of 0.8 mL/min, and an oven temperature of 45 �C.

2.5. Microbiological studies

Samples (w25 mL) of suspended solids for microbiological

studies were taken during the operation of the reactor. Batch

tests and MPN culturing were performed immediately. For

fluorescence in situ hybridisation analysis (FISH) and DNA

extraction, the samples were centrifuged for 15 min at 6000 g

and 4 �C. The supernatant fractions were decanted, and the

cell pellets were stored at �20 �C for DNA extraction or fixed

with paraformaldehyde [19] and then stored at �20 �C for

FISH.

The hydrogen production capacity was determined in

batch experiments measuring the specific hydrogen activity

as previously described [20]. The substrate was the same

waste product used for feeding the reactor in a final concen-

tration of 1000 mgCOD/L.

In specified samples (day 175 and 247), the presence of cells

from the domain Archaea were determined by FISH using the

Arc 915 probe [19] as previously described [21].


Samples taken on operation day 69 and 195 were used to

determine the number of hydrogen-producing bacteria by the

MPN method. Anaerobic medium supplemented with glucose

(Sigma, 10 g/L), triptone (Difco, 5 g/L), yeast extract (Difco, 5 g/

L) and meat extract (5 g/L) was used. The pH was adjusted to 5

with HCl, and bromocresol purple was used as the pH indi-

cator. The media was sparged in a N2 atmosphere and steri-

lised in an autoclave. After inoculation, tubes were incubated

at 30 �C for 24–76 h. Cultures were considered positive for

hydrogen-producing bacteria when visible growth and

hydrogen in the gas phase (measured by GC) were present.

Positive cultures from the highest dilutions of the sample

taken at day 69 were used to obtain isolates. Serial dilutions (1/

10) were performed in the same medium until only one

morphology was detected by microscopic observation using

Gram staining. Isolation was then performed in roll tubes

using the same anaerobic medium solidified with 2% agar

(Difco). Single colonies were picked and transferred to the

same liquid medium. The purity of the culture and the

morphology of the isolates were tested using Gram staining.

Isolates were characterised by 16S rRNA gene sequence anal-

ysis. DNA was extracted from liquid cultures using the DNA

Wizard extraction kit (Promega), carried out according to the

manufacturer’s suggestions for Gram positive bacteria. The

16S rRNA gene was amplified by PCR using the universal

Bacteria primers (forward 27F: 50-AGAGTTTGATCCTGGCTC

AG-30, corresponding to positions 8� 27 using Escherichia coli

numbering; reverse1522R 50-AAGGAGGTGATCCAGCCGCA-30,

corresponding to positions 1522� 1542). Amplification reac-

tions were performed as described in [22]. PCR product puri-

fication and sequencing were performed by Macrogene Inc.

(Korea) Sequencing service (Korea).

The utilisation of various substrates by the isolates was

determined using a basal anaerobic medium containing yeast

extract (Difco; 1 g/L) [22] supplemented with glucose (Sigma),

or sodium lactate (Aldrich) (10 mM). Growth was measured

spectrophotometrically (Genesys 5; Spectronic, Milton Roy) at

660 nm. Fermentation products (H2 and volatile fatty acids)

were determined for each different substrate as described.

Experiments were performed in triplicate.

The microbial community composition was studied

by Terminal Restriction Fragment Length Polymorphism

(T-RFLP) of the 16S rRNA present in the samples taken on the

following reactor operation days: 0, 69, 175 and 247. DNA was

extracted using an UltraClean Soil DNA Isolation Kit (MO BIO

Laboratories Inc.) according to the manufacturer’s protocol.

The 16S rRNA genes were amplified by PCR using the same

Bacteria universal primers used previously, but the forward

primer was fluorescently labelled with the dye 6-FAM (5-[6-

carboxy-fluorescein]). The amplification reaction was carried

out as described in [23]. The amplification products were

purified using a PCR purification kit (QIAGEN, Courtaboeuf,

France), and digested with the Msp I restriction enzyme (Fer-

mentas) according to the manufacturer’s suggestions. After

enzyme inactivation by heat treatment (65 �C for 1 h), DNA

fragments were precipitated with 90% ethanol and washed

twice in 70% ethanol. DNA fragments were dried at 65 �C and

then re-suspended in 8 mL formamide and 0.3 mL of internal

standard (GeneScan-500 Liz Standard, Applied Biosystems).

The terminal restriction fragments (T-RF) were separated on

an ABI3130 Genetic Analyzer (Applied Biosystems) at the

Molecular Biology Unit (Institut Pasteur – Montevideo). Chro-

matograms were analysed and manually aligned using Peak

Scanner Software v. 1.0 (Applied Biosystems). To avoid primer

artefacts, fragments smaller than 50 bp were not included, and

the peak heights were standardised to the minimum sample as

described previously [24]. The alignment resulted in a matrix,

where each peak was considered indicative of a different gene

and peak heights were used as a measurement of gene abun-

dance. The relative abundances of T-RF were determined by

calculating the ratio between the height of each peak and the

sum of all peak heights within each sample.

To identify the predominant peaks in the T-RFLP, a 16S

rRNA gene clone library was constructed for the sample taken

at operation day 69. The 16S rRNA genes were amplified as

previously described, using unlabelled primers. The PCR

products were cloned using the TOPO TA cloning kit for

sequencing (Invitrogen) according to the manufacturer’s

instructions. Colonies were chosen randomly, and sequences

from the plasmid insert were determined using the forward

primer of the cloned gene. DNA sequencing was conducted

using an ABI Prism 3700 gene analyzer (Applied Biosystems) at

the Michigan State University Genomics Technology and

Support Facility.

Sequences from clones and isolates were compared with

sequences from the NCBI database using Blastn Search (nucle-

otide–nucleotide comparison) and from the Ribosomal Data-

base Project (RDP) using the Classifier Tool. Clones having a 16S

rRNA sequence similarity of more than 97% with each other

were grouped into an operational taxonomic unit (OTU).

Representative sequences were selected and aligned to related

sequences from the NCBI database with ClustalW, and a phylo-

genetic tree was constructed using MEGA version 3.1 [25]. Seq-

boot was used to obtain the confidence level in 500 datasets.

Sequences were digested ‘‘in silico’’ with the enzyme used

for T-RFLP to compare the T-RFLP peaks with the sequences

retrieved from strains and clones. The number of nucleotides

of the 50 ‘‘in silico’’ fragments were determined and compared

to the T-RF lengths.

3. Results and discussion

3.1. pH monitoring

After recording a significant drop in the reactor pH to 3.3

after 5 days of operation, the cheese whey feedstock was

supplemented with NaHCO3 to increase its alkalinity level

(0.2 gNaHCO3/gCOD). After pH recovery (around day 20), the

pH at the outlet was always greater than 4. The average pH at

the inlet and at the outlet of the reactor was 5 with a standard

deviation of 1 (49 samples). The observed pH variation at the

inlet over time may be explained by partial fermentation of

the whey, although it was maintained at 4 �C until used.

3.2. Hydrogen production

The reactor operation started with a hydraulic residence time

(HRT) of 24 h and an organic loading rate of 10 gCOD/L.d

(Table 1). Under these conditions, the biogas production was

Table 1 – Operating conditions.

Day HRT (h) VSS reac (g/l) OLR (gCOD/l.d) OLR (gCOD/gVSS.d) Range of methanecontent of the biogas (%)

Range of hydrogencontent of the biogas (%)

0–56 24 7–16 10 0.6–1.4

57–99 12 11–16 20 1.2–1.8 6–12 <1

100–139 24 10–18 10 0.6–1.0 9–19 <1

140–219 12 12–17 20 1.2–1.6 15–20 <1

220–260 12 7–9 20 2.2–2.8 2–5 20–30


very unstable with an important presence of methane (10–

20%) and almost no H2 production (<1%). In order to inhibit

methane production, the HRT was reduced to 12 h on day 56,

but hydrogen production was still very low and unstable. Due

to an operational problem on day 100, the biomass was

washed from the reactor. Then, to favour biomass growth,

HRT was increased to 24 h for 40 days. After biomass recu-

peration, the HRT was again set at 12 h (day 140). Biogas

production remained unstable and methane was still present.

The concentration of biomass in the reactor was not stable

because of its significant growth. To solve this problem, from

day 220 onward the purge regime was set at 0.5 gSS/d. In these

new conditions, the biomass concentration and the biogas

production stabilised to values of 7–8 gVSS/L and 2 L/d,

respectively. Hydrogen content in the biogas increased to

20–30%. Methane was still present, but in a lower concentra-

tion (<5%) (Fig. 1). Table 1 shows the operating conditions

throughout the operational period. An average hydrogen

production of 550 mL H2/d (or 122 mL H2/Lreac d) was obtained

for an organic loading rate of 20 gCOD/L.d and 2.5 gCOD/

gVSS.d (average value). The overall production obtained for

the entire operating period was 122 mL H2/L.d.

Increasing the solids purge regime stabilised the solids

concentration in the reactor. The resulting increase in

hydrogen production could be attributed to the increase in the

organic loading rate per grams of volatile suspended solids.

Nevertheless, hydrogen production was very low compared to

previously published values. Yang et al. [9] reported approxi-

mately 0.8 LH2/L.d for a loading rate of 10 gCOD/L.d and a HRT

of 24 h using a CSTR. Yang et al. also detected methane

production below pH 5 showing, as in the present work, the

possibility of methane production in acidic conditions.

However, Davila-Vazquez and colleagues [10] also used

cheese whey as a substrate, and did not detect methane

0200400600800

1000120014001600

0 50 100 150 200 250 300Time (d)

Meth

an

e an

d H

yd

ro

gen

p

ro

du

ctio

n (m

l/d

)

MethaneHydrogen

Fig. 1 – Methane and hydrogen production during reactor’s

operation.

production even at pH 7.5. Other authors [14] working with

a UASB fed with sucrose (HRT 8 h, 20 gCOD/L) obtained

6.7 LH2/L.d without production of methane.

More research is needed to determine the effect of adding

nutrients to the whey in order to increase hydrogen produc-

tion. In previously reported works, the substrate was supple-

mented with macro- and micronutrients. In this work,

unsterilised cheese whey was used to feed the reactor without

any nutrient addition. This alternative has the advantage of

lower costs of operation, but the negative effect on hydrogen

production should be evaluated.

3.3. VFA production

Figs. 2 and 3 show the results of the volatile fatty acid (VFA)

analysis of liquid samples taken at the inlet and outlet of the

reactor throughout the operation. According to these data, the

cheese whey was partially fermented prior to its entrance into

the reactor (Fig. 2): approximately 20% of the whey COD cor-

responded to lactic acid. At the outlet of the reactor, the VFAs

were composed mostly of propionic, acetic and low amounts

of valeric acid (Fig. 3). The lactic acid concentration at the

outlet was very low, indicating that it was converted mostly to

propionic and acetic acid. During the last operating period

(after day 220), the propionic acid concentration in the effluent

decreased, while the concentrations of butyric and acetic

acids increased. This change is consistent with the observed

increase in H2 production [1].

3.4. Biomass characteristics

During reactor operation, the biomass presented good sedi-

mentation characteristics with a very high VSS content (more

than 90%). Sludge granulation was not evident, the biomass

became white and small aggregates (less than 1 mm) were

observed. Similar biomass characteristics including small

sized aggregates, whitish colour and with low amounts of ash,

have been reported by other authors [14,26]. However, these

studies also showed the presence of small granules. It has

been proposed that the white colour and low ash content may

be due to wash-out of sulphate-reducing bacteria with the

concomitant loss of inorganic sulphide precipitation [26].

3.5. Hydrogen specific activity and FISH

The specific activity values of hydrogen in the sludge samples

(Fig. 4) were found to be quite variable, with values ranging

between 300 and 1900 mmolH2/day.gVSS. Although batch tests

were performed at pH 5, the hydrogen activity could not be

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

13 29 41 50 57 69 78 97 113

125

134

202

216

225

240

254

275

293

Time (d)

VFA

(m

gC

OD

/l)

Fig. 2 – Volatile fatty acids at the inlet of the reactor.


determined for some samples due to hydrogen-consuming

methanogenesis. In all samples tested, cells hybridising with

the Archaea probe were detected by FISH, indicating that the

methanogens were not completely washed out of the reactor

in spite of the low pH during operation. These results indicate

that it is possible to produce methane at low pH. Although

most of the ‘‘known’’ methanogens could not grow at low pH,

it has been reported that some isolates from the genera

Methanosarcina and Methanobacterium were capable of growing

and producing methane at pH values of 5.0 and 4.68, respec-

tively [27,28]. Other reports on methanogenesis at low pH

confirmed these findings [29]. Jain and Mattiasson reported

methane production at pH values of 6.0, 5.5, 5.0, and 4.5, and

4.0 using activated sludge as seed [30]. To achieve acidic

conditions, they lowered the pH step-wise from 7.0 to pH 4.0 in

increments of 0.5. Initially, a decrease in methane production

was observed, but after some acclimatisation, methane

production returned to levels observed at higher pH values.

Therefore, the acclimatisation of methanogens to low pH

could explain the production of methane in our process as

well. Other parameters such as the organic loading rate or the

HRT should be controlled to produce a selective wash-out of

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

13 29 41 50 57 69 78 97 1Ti

VFA

(m

gC

OD

/l)

Fig. 3 – Volatile fatty acids at

the methanogens in this kind of hydrogen-producing reactor.

Gavala et al. [31] did not detect methane in the biogas during

operation of a UASB at pH values of 4.4–4.5, without pH

control. That study utilised glucose as the substrate, operated

the reactor with three low HRTs (12, 6 and 2 h), and employed

a mixture of granular sludge sterilised three times by auto-

clave with sludge from a CSTR hydrogen-producing reactor as

the inoculum. The differences between those results and the

present experiments could be explained either by the inoc-

ulum source, the use of synthetic sterilised wastewater or by

the operation at low HRT. More research is needed to deter-

mine which of these parameters is determinant for hydrogen

production in upflow anaerobic sludge bed reactors.

3.6. Hydrogen-producing bacteria count (MPN)and isolation

Hydrogen-producing bacteria were detected in high numbers

by the MPN technique (at day 69, >2.4� 1011 MPN/mL and at

day 195, 9.0� 1011 MPN/mL). The predominant organisms

were isolated from diluted cultures obtained from the MPN

assay. Two strains (H1 and H2) were isolated under anaerobic

13 125 134 202 216 225 240 254 275 293me (d)

the outlet of the reactor.

0

500

1000

1500

2000

2500

0 50 100 150 200 250 300Day of operation

SH

A

(µm

ol H

2/g

SS

V.d

ay)

SHAMethane presence

Fig. 4 – Specific hydrogen activity determined in sludge

samples taken at different reactor operation days in batch

experiments.


conditions; both were Gram positive with short rod

morphology. Both produced hydrogen, propionic acid and

acetic acid during glucose fermentation. The isolates also

utilised lactic acid as a substrate, producing only acetic and

propionic acid (no hydrogen was detected).

Phylogenetic analysis of the 16S rRNA gene sequences

(Fig. 5) showed high homology (99%) with Pectinatus portalensis,

a fermenting microorganism isolated from a wastewater

treatment plant [32].

Fig. 5 – Phylogenetic tree showing the affiliation of the 16S

rRNA gene sequences from the clone library and from the

isolates. The Neigbour-Joining tree was constructed using

429 nucleotide positions. Sequences retrieved from the

NCBI database were included (the accessing numbers are

included in brackets) and Methanococcus aeolicus

(DQ195964) was used as out-group. Sequences from this

work were deposited in the database (NCBI) with the

following accessing numbers: otu1: FJ668019; otu 2:

FJ668020; otu3: FJ668021; otu4: FJ668022; otu5: FJ668023;

otu6: FJ668024; otu7: FJ668025; otu8: FJ668026; otu9:

FJ668027; strain H1: FJ668028; strain H2: FJ668029. The

scale bar represents five nucleotide substitutions per 100

nucleotides. Bootstrap values (500 replicates) above 50%

are shown at branch nodes.

3.7. T-RFLP and 16S rRNA gene library

The total microbial population was studied by T-RFLP of the

16S rRNA genes. As shown in Fig. 6, the bacterial community

changed during the course of reactor operation. Particularly,

an increase in the T-RFs of 91, 129, 164, 300, and 383 nucleo-

tides was detected over time, while the T-RF of 177 nucleo-

tides was detected at the beginning but less abundantly in the

last two samples.

In order to identify the predominant organisms in the

community, a 16S rRNA gene library was constructed for the

sample taken at day 69. A total of 84 clones were sequenced,

and the sequences grouped into 9 OTUs (Table 2). Sequence

comparison with a database (RDP) and phylogenetic analysis

revealed a high abundance of organisms from the Firmicutes

phylum and relationships to several genera with sugar

fermentation capacity (Table 2 and Fig. 5).

Sequences from the library were correlated with the T-

RFLP peaks according to the ‘‘in silico’’ digestion. Two of the T-

RFLP peaks that increased during reactor operation correlated

to sequences from the genera Anaerotruncus (T-RF 91), Mega-

sphaera and Mitsoukella (T-RF 300–301) (Table 2). Hydrogen

production has been previously reported for species of the

genera Anaerotruncus, Megasphaera and Pectinatus (Table 2);

thus, their presence in the community could explain the

observed production of hydrogen in the present study.

Species of the genus Lactobacillus (T-RF 177) produce lactic

acid by fermentation of sugars [41]. Lactobacilli are frequently

found in cheese-producing facilities, as they are used in

several fermentation processes [42]. Thus, their presence was

expected in a reactor fed with unsterilised cheese whey. Most

species of Lactobacillus are not hydrogen producers, but

recently, Yang et al. [9] reported the production of high

quantities of H2 by a Lactobacillus strain (rennanqilfy16) iso-

lated from a H2-producing reactor. Accordingly, the H2

production capacity of the genus Lactobacillus should be re-

evaluated.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

day 0 day 69 day 175 day 247Reactor operation day

Relative ab

un

dan

ce

553548436383300296236177164129979168

Fig. 6 – 16S rRNA gene T-RFLP community analysis during

reactor operation.


Lactic acid was detected in low amounts at the reactor

outlet, indicating that lactose fermentation was not the

predominant pathway inside the reactor. Moreover, lactic acid

was consumed during the reactor operation. It was previously

reported that members of the Pectinatus [43] and Megasphaera

[33] genera are able to consume lactic acid, producing propi-

onate. This property was also detected in our isolates; there-

fore, the presence of these organisms may explain the lactic

acid consumption.

Although two strains from the genus Pectinatus were iso-

lated, this organism was not dominant in the T-RFLP or in the

clone library. It was detected in low proportion by both

methods (only one clone was detected in the library and the

abundance of the T-RFLP peak was less than 1% (data not

shown). This result clearly shows the bias of the methods

used; thus, the importance of this organism in the total

biomass should be evaluated by other methods, such as FISH

or quantitative PCR.

Several genera of fermenters (Prevotella, Olsenella, Bulleidia,

Mitsoukella and Selenomonas) with no fermentative hydrogen

production reported in the literature were detected in the

Table 2 – Characterization of the clone library and isolates accodatabase and main fermentation products reported in the biblithe clones and strains 16S rRNA gene sequences were also shchromatograms, for that correlation a tolerance of D/L 2 base

Generaa Main fermentation

otu 1 Prevotella (76%) Acetic, Lactic

otu 2 Olsenella (56%) Acetic, Lactic

otu 3 Bulleidia (82%) Acetic, Lactic

otu 4 Anaerotruncus (61%) H2, Acetic, Butyric

otu 5 Mitsuokella (76%) Acetic, Lactic, Succinic

otu 6 Selenomonas (94%) Acetic, Lactic (not all),

otu 7 Megasphaera (100%) H2 (not all), Acetic, Pro

otu 8 Megasphaera (100%) H2 (not all), Acetic, Pro

otu 9 Lactobacillus (100%) H2 (not all), Lactic

Isolates H1 and H2 Pectinatus (100%) H2, Acetic, Propionic

a The genera and phylum were determined using the RDP classifier tool.

b Fermentation products from sugars in representatives from the genera

c Predicted T-RFs length (in nucleotides) were determined from the sequ

biomass by both T-RFLP and the clone library. Members of

these genera were also detected in other hydrogen production

reactors [44,45] suggesting that these organisms could out-

compete the hydrogen-producing organisms, thereby

decreasing the hydrogen yield. Thus, to improve H2 produc-

tion it will be necessary to study the physiology of these

organisms to find the optimal reactor operation conditions to

avoid their growth.

4. Conclusions

The results from this work demonstrated the feasibility of

producing hydrogen in a UASB reactor using unsterilised

cheese whey as the substrate. However, under the operational

conditions tested, hydrogen production was low (122 mL H2/

L.d for an OLR of 20 gCOD/L.d and 2.5 gCOD/gVSS.d). These

results are very important because they demonstrate the

applicability of the process using raw waste material.

As reported in the literature, as well as in our work, several

factors influence hydrogen production in continuous biore-

actors (seed material, pH of operation, HRT, OLR, and so forth).

Our results show that operational pH is an important

parameter to promote H2 production, but it is not sufficient to

control methane production. The biomass appeared to accli-

matise to the low pH, as methanogenesis was restored even at

pH 5.

It was also shown that the use of a high loading rate per

gram of VSS and low HRT favoured hydrogen production in

reactors with biomass retention. More studies should be done

in order to find the optimal values for these parameters in

UASB reactors for hydrogen production from industrial

wastewaters.

In order to better understand the process, the microbial

community evolution was studied during the operation and

linked to the reactor performance. Organisms belonging to the

genera Anaerotruncus, Megasphaera and Pectinatus were detec-

ted by both culture and non-culture methods (T-RFLP, 16S

rRNA cloning library and isolation); these organisms may be

rding to the 16S rRNA gene sequence comparison with RDPography for these genera. The predicted T-RF calculated forown, in bold are marked the T-RFs detected in the T-RFLPs was assume.

productsb Reference Predicted T-RF length (nt)c

[34] 97

[35] 169

[36] 162

[37] 90

[38] 300

Propionic [39] 295

pionic [33,40] 301

pionic [33,40] 197

[41,9] 177

This work, [43] 291

The % represents the probability according to the RDP classifier tool.

according to the bibliography.

ences by ‘‘in sillico’’ digestion using the enzyme Msp I.


responsible for the hydrogen production. The low hydrogen

yield could be explained by the presence of fermentative

organisms with low yield (such as the propionate producers

Megasphaera and Pectinatus) and by fermenters unable to

produce hydrogen that could have competed for the substrate

(such as Prevotella, Olsenella, Bulleidia, Mitsoukella and Seleno-

monas). It was also shown that the methanogenic population

could not be inhibited or washed out from the reactor, which

is another factor that could explain the low hydrogen yield.

In order to improve the hydrogen yield in this type of

reactor, it will be necessary to perform more studies to find the

optimal operating conditions to enrich hydrogen fermenting

organisms (such as Clostridium butyricum) while avoiding

hydrogen consumers. It is also important to continue inves-

tigating the use of unsterilised industrial waste products to

observe the effects of the incoming organisms present in the

reactor feedstock.

Acknowledgements

The authors want to thank Conaprole (milk processing factory

located in San Ramon, Canelones, Uruguay) for submitting the

cheese whey weekly and the Center for Microbial Ecology

from Michigan State University for the sequencing assistance.

This work was financed by DINACYT (Uruguay), PDT 47/15.

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